Methods for Diagnosing Pervasive Development Disorders, Dysautonomia and Other Neurological Conditions

ABSTRACT

Methods for aiding in the diagnosis of disorders including, but not limited to, PDDs (Pervasive Development Disorders), Dysautonomic disorders, Parkinson&#39;s disease and SIDS (Sudden Infant Death Syndrome). In one aspect, a diagnosis method comprises analyzing a stool sample of an individual for the presence of a biological marker (or marker compound) comprising one or more pathogens, which provides an indication of whether the invidual has, or can develop, a disorder including, but not limited to, a PDD, Dysautonomia, Parkinsons disease and SIDS. Preferably, the presence of one or more pathogens is determined using a stool immunoassay to determine the presence of antigens in a stool sample, wherein such antigens are associated with one or more pathogens including, but not limited to,  Giardia, Cryptosporidium, E. histolytica, C. difficile , Adenovirus, Rotavirus or  H. pylori.

CROSS REFERENCE TO RELATED APPLICATION

This application is based on, and claims the benefit of, U.S.Provisional Application No. 60/249,239, filed on Nov. 16, 2000, which isfully incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention generally relates to methods for aiding in thediagnosis of disorders including, but not limited to, PDDs (PervasiveDevelopment Disorders), Dysautonomic disorders, Parkinsons disease andSIDS (Sudden Infant Death Syndrome). More particularly, the inventionrelates to a diagnosis method comprising analyzing a stool sample of anindividual for the presence of a biological marker (or marker compound)comprising one or more pathogens, which provides an indication ofwhether the invidual has, or can develop, a disorder including, but notlimited to, a PDD, a Dysautonomic disorder, Parkinson's disease or SIDS.

BACKGROUND

Currently, extensive research is being conducted to determineassociations between gastrointestinal dysfunction and a variety of humandisorders that, heretofore, have been of unknown etiology. For example,an association between dysautonomic conditions and gastrointestinaldysfunction has been described in U.S. patent application Ser. No.09/929,592, filed on Aug. 14, 2001, entitled “Methods For Diagnosing andTreating Dysautonomia and Other Dysautonomic Conditions, which iscommonly owned and fully incorporated herein by reference. Further, arelationship between gastrointestinal conditions and PDDs such asAutism, ADD (Attention Deficit Disorder) and ADHD (Attention DeficitHyperactivity Disorder) has been described in detail in U.S. patentapplication Ser. No. 09/466,559, filed Dec. 17, 1999, entitled “MethodsFor Treating Pervasive Development Disorders,” and U.S. Ser. No.09/707,395, filed on Nov. 7, 2000, entitled “Methods For TreatingPervasive Development Disorders”, both of which are commonly owned andincorporated herein by reference.

Based on these findings, it is thus desirable to continue research infinding biologic markers of gastrointestinal dysfunction that may aid inthe diagnosis of certain diseases and disorders. For example, the effectof various pathogens on the gastrointestinal tract, and the associationof such pathogens to disorders such as PDD and dysautonomia, hasheretofore not been researched. Various microorganisms that are ofinterest will now be discussed.

Helicobacter pylori (H. pylori) is generally associated with chronicgastritis and peptic ulcer in children and adults. The prevalence of H.pylori is highest in developing countries and lowest in developedcountries. Ethnicity, socioeconomic status, household crowding, andother conditions contribute to the formation of H. pylori infection.Infection is rarely symptomatic in children, and duodenal ulcers aregenerally not seen in children less than 10 years of age. Variousdiseases that are caused, or believed to be caused by H. pyloriinfection are known. For instance, it has been postulated that H. pyloriplays a role in auto-immune athero-sclerosis.

Esophageal reflux disease (GORD) has further been postulated to becaused by H. pylori in a mechanism whereby somatostatin induces thehypothalamus to decrease the release of growth hormone from thepituitary affecting the adrenal control of cortisol. The change incortisol ultimately affects the gastrin release mechanism causing anincrease in acid.

Cryptosporidium parvum can be associated with infections of thegastrointestinal tract in children and in immunocompromised populations.It is generally thought to account for up to 20% of all cases ofdiarrhea in developing countries and potentially life threatening inchildren with AIDS due to the induction of severe malnutrition. Theseinfections are generally asymptomatic and occur in tandem with otherinfections such as one with Giardia.

In 1993, a large outbreak of Cryptosporidium parvum occurred inMilwaukee Wisconsin in which 400,000 people were affected. It has aseasonal effect of being more prevalent in the late summer in childrenunder the age of 15 years.

Giardia lamblia is a common cause of diarrhea in humans and othermammals throughout the world. In its most severe form, it has been foundto cause infectious lymphocytosis. Although rare, infection with Giardiacan be protracted and debilitating. Giardia lamblia is a flagellate thatencysts, and generally does not cause symptomotology. However, whenfound in the trophozoite form, severe diarrhea can result. Symptoms caninclude diarrhea, vomiting, fatigue, and growth retardation in children.Malabsorption results from infection with the trophzoite form, andpotential blockage of the microvilli of the intestines occurs. There maybe an interaction between decreased levels of IgA in thegastrointestinal system and giardiasis.

Clostridium infections of the gastrointestinal tract are of theperfringes, botulinum and difficele varieties. Perfringens foodpoisoning is the term used to describe the common foodborne illnesscaused by C. perfringens. A more serious but rare illness is also causedby ingesting food contaminated with Type C strains. The latter illnessis known as enteritis necroticans. The common form of perfringenspoisoning is characterized by intense abdominal cramps and diarrheawhich begin 8-22 hours after consumption of foods containing largenumbers of those C. perfringens bacteria capable of producing the foodpoisoning toxin. The illness is usually over within 24 hours but lesssevere symptoms may persist in some individuals for 1 or 2 weeks. A fewdeaths have been reported as a result of dehydration and othercomplications. Necrotic enteritis caused by C. perfringens is oftenfatal. This disease also begins as a result of ingesting large numbersof the causative bacteria in contaminated foods. This disease is a foodinfection; only one episode has ever implied the possibility ofintoxication (i.e., disease from preformed toxin).

Clostridium difficile is an infection generally caused by changes in theintestinal mucosa. Those changes are caused by an overuse of antibioticscreating an intestinal environment favorable to the infiltration withClostridium difficile. Infection with C. difficile is generallydebilitating and C. difficile is a gram-positive, spore forming,anaerobic bacillus which can produce toxin-mediated diarrhea orpseudomembranous colitis. It has been isolated from soil, sand, hay, andanimal dung. C. difficile colonization of the colon occurs in 2%-3% ofhealthy adults. Following exposure to antibacterial agents, the rate ofasymptomatic colonization in adults averages between 5% to 15%, butrates as high as 46% have been reported. Carriage rates of up to 70%have been reported in children below the age of one year, but by twoyears of age the “normal” colonic flora is established and the frequencyof colonization decreases to that of healthy adults. Of interest is thathealthy children less than one year of age are the only population inwhich C. difficile toxins are frequently detected in the stool in theabsence of clinical symptoms. One suggestion advanced to explain thisobservation is that the infant's gut cannot respond to the toxin.

Clostridium botulinum is an anaerobic, spore-forming rod that produces apotent neurotoxin. The spores are heat-resistant and can survive infoods that are incorrectly or minimally processed. Seven types (A, B, C,D, E, F and G) of botulism are recognized, based on the antigenicspecificity of the toxin produced by each strain. Types A, B, E and Fcause human botulism. Types C and D cause most cases of botulism inanimals. Animals most commonly affected are wild fowl and poultry,cattle, horses and some species of fish. Although type G has beenisolated from soil in Argentina, no outbreaks involving it have beenrecognized. Foodborne botulism (as distinct from wound botulism andinfant botulism) is a severe type of food poisoning caused by theingestion of foods containing the potent neurotoxin formed during growthof the organism. The toxin is heat labile and can be destroyed if heatedat 80oC for 10 minutes or longer. The incidence of the disease is low,but the disease is of considerable concern because of its high mortalityrate if not treated immediately and properly. Most of the 10 to 30outbreaks that are reported annually in the United States are associatedwith inadequately processed, home-canned foods, but occasionallycommercially produced foods have been involved in outbreaks. Sausages,meat products, canned vegetables and seafood products have been the mostfrequent vehicles for human botulism.

The life cycle of Entamoeba histolytica involves trophozoites (thefeeding stage of the parasite) that live in the host's large intestineand cysts that are passed in the host's feces. Humans are infected byingesting cysts, most often via food or water contaminated with humanfecal material. The trophozoites can destroy the tissues that line thehost's large intestine, so of the amoebae infecting the humangastrointestinal tract, E. histolytica is potentially the mostpathogenic. In most infected humans the symptoms of “amoebiasis” (or“amebiasis”) are intermittent and mild (various gastrointestinal upsets,including colitis and diarrhea). In more severe cases thegastrointestinal tract hemorrhages, resulting in dysentery. In somecases the trophozoites will enter the circulatory system and infectother organs, most often the liver (hepatic amoebiasis), or they maypenetrate the gastrointestinal tract resulting in acute peritonitis;such cases are often fatal. As with most of the amoebae, infections ofE. histolytica are often diagnosed by demonstrating cysts ortrophozoites in a stool sample. Infections that sometimes last for yearsmay be accompanied by no symptoms, vague gastrointestinal distress,and/or dysentery (with blood and mucus). Most infections occur in thedigestive tract but other tissues may be invaded. Complications includeulcerative and abscess pain and, rarely, intestinal blockage. Onset timeis highly variable. It is theorized that the absence of symptoms ortheir intensity varies with such factors as strain of amoeba, immunehealth of the host, and associated bacteria and, perhaps, viruses. Theamoeba's enzymes help it to penetrate and digest human tissues; itsecretes toxic substances.

No extensive research is known to have been conducted heretofore todetermine correlations and associations regarding the presence ofpathogens in the gastrointestinal tract of individuals in, e.g., PDD,Parkinson's and Dysautonmia populations. Based on the findings describedherein in accordance with the present invention, correlations andassociations are found to exist between various disorders such asAutism, Parkinson's, ADD, ADHD and Dysautonomia, for example, and thepresence of pathogens in an individual's digestive tract.

SUMMARY OF THE INVENTION

The present invention generally relates to methods for aiding in thediagnosis of disorders including, but not limited to, PDDs, Dysautonomicdisorders, Parkinson's disease and SIDS. More particularly, theinvention relates to a diagnosis method comprising analyzing a stoolsample of an individual for the presence of a biological markercomprising one or more pathogens, which provides an indication ofwhether the invidual has, or can develop, a disorder including, but notlimited to, a PDD, Dysautonomia, Parkinson's disease and SIDS. In apreferred embodiment, a stool immunoassay is used to determine thepresence of antigens in a stool sample, wherein such antigens areassociated with one or more pathogens including, but not limited to,Giardia, Cryptosporidium, E. histolytica, C. difficile, Adenovirus,Rotavirus or H. pylori.

In another aspect of the invention, pathogens including, but not limitedto, Giardia, Cryptosporidium, E. histolytica, C. difficile, Adenovirus,Rotavirus, and H Pylori, comprise biological markers whose presence in astool sample, for example, are efficacious for determing whether anindividual, especially a child, has, or can potentially develop, adisorder including, but not limited to, a PDD, Dysautonomia, Parkinsonsdisease, SID, and/or other neurological disorders.

These and other aspects, features, and advantages of the presentinvention will be described and become apparent from the followingdetailed description of preferred embodiments, which is to be read withthe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table diagram illustrating various pathogens that testedpositive in stool samples of individuals having a Dysautonomic disorder;

FIG. 2 is a table diagram illustrating various pathogens that testedpositive in stool samples of individuals having Parkinson's, as comparedwith stool results of individuals not having Parkinson's disease;

FIG. 3 is a table diagram illustrating various pathogens that testedpositive in stool samples of individuals having ADD or ADHD, as comparedwith stool results of individuals not having ADD or ADHD; and

FIG. 4 is a table diagram illustrating various pathogens that testedpositive in stool samples of individuals having a PDD.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is directed to methods for aiding in the diagnosisof various human disorders, such as PDD, Dysautonomia, Parkinson's,SIDS, etc. In one aspect, a method comprises analyzing stool samples ofan individual to determine the presence of pathogens including, but notlimited to, Giardia, Cryptosporidium, Entamoeba histolytica, Adenovirus,Rotavirus, H pylori, Cyclospora, Microsoridia, and/or Isospora belli.Preferably, the presence of one or more pathogens is determined using astool immunoassay to determine the presence of antigens in a stoolsample, wherein such antigens are associated with one or more pathogensincluding, but not limited to, Giardia, Cryptosporidium, E. histolytica,C. difficile, Adenovirus, Rotavirus or H. pylori.

In particular, a stool immunoassay results in the determination of thepresence of a particular antigen (usually a protein) that the particularpathogen leaves behind (i.e., each microorganism is associated with aspecific antigen). This antigen represents a pathogen, and is recognizedby the gastrointestinal tract of the individual as a foreign protein. Inaccordance with one aspect of the invention, the presence of one or moreantigens, regardless of the quantitative level, comprises a biologicalmarker for determining if the person, especially a child, may eitherhave or develop a disorder such as a PDD, Dysautonomia, Parkinson's, orSIDS.

Until now, there has been no known methods for analyzing stool samplesto determine the presence of pathogens as biological markers to allowearly diagnosis or screening of such disorders or conditions. It ispostulated, for example, that the presence of antigen(s) and/or themicroscopic presence of such organisms may signal the formation of adysbiosis, and ultimately the formation of a malabsorption syndrome.This malabsorption syndrome can predispose the individual to theformation of a disorder such as autism, ADD, ADHD, SIDS, PDD, tourettes,OCD and other neurological conditions. In particular, the formation ofmalabsorption syndrome can affect, for instance, proper and essentialprotein digestion/absorption. And in the absence of proper proteindigestion/absorption, the amino acids necessary for, e.g., the growthand development or normal functioning of certain chemical processes ofindividuals are absent.

Consequently, it is postulated, for example, that a lack of properprotein absorption, for instance, of an individual, especially children,can lead to various disorders such as autism, other PDDs, SIDS, andother disorders mentioned herein. Indeed, as described in each of theabove-incorporated U.S. Patent Applications, abnormal protein digestionis found to occur in the PDD and dysautonomic populations. For instance,it was further determined that a sub-population of individuals sufferingfrom ADD, ADHD and autism as well as a sub-population of those withdysautonomic conditions had an abnormal level of the enzymechymotrypsin, indicated pancreatic insufficiency as a component of suchdisorders.

The following case studies indicate that there are correlations betweenthe development of various disorders and the presence of microorganismsin an individual's digestive tract. It is to be understood that theseexamples are set forth by way of illustration only, and nothing thereinshall be taken as a limitation upon the overall scope of the invention.

Case 1:

Stool sample were collected from two children diagnosed as havingFamilial Dysautonomia. The stool samples were analyzed for the presenceof pathogens. As shown by the table in FIG. 1, the stool sample of Child1 tested positive for H. pylori, Giardia, and Cryptosporidium. Further,the stool sample of Child 2 tested positive for H. pylori, Giardia,Cryptosporidium and Rotavirus.

Case 2:

Stool samples were collected from 15 individuals diagnosed as havingParkinson's disease. The stool samples were analyzed for the presence ofpathogens. Further, stool samples were collected from an additional 15individuals who were not diagnosed as having Parkinson's disease, norhaving known familial association with Parkinson's or known GIconditions. These stool samples were also analyzed for the presence ofpathogens.

The table in FIG. 2 illustrates the result of this study. As shown, thestools of each of the 15 individuals diagnosed as having Parkinson'sdisease tested positive for various pathogens including H. pylori,Cryptosporidium, E. hystolytica, Giardia, Rotavirus, Camphylobacter,and/or C difficile. On the other hand, virtually all the stools of eachof the 15 individuals not diagnosed as having Parkinson's disease testednegative for such pathogens.

Case 3:

Stool samples were collected from 13 children diagnosed as having eitherADD or ADHD and analyzed for the presence of pathogens. Further, stoolsamples were collected from an additional 14 children not diagnosed ashaving ADD or ADHD and analyzed for the presence of pathogens.

The table in FIG. 3 illustrates the result of this study. As shown, thestools of each of the 13 children diagnosed as having either ADD or ADHDtested positive for various pathogens including as H. pylori,Cryptosporidium, E. hystolytica, Giardia, Rotavirus, Camphylobacter,and/or C difficile. On the other hand, virtually all the stools of eachof the 14 children not diagnosed as having ADD or ADHD tested negativefor such pathogens.

Case 4:

Stool samples were collected from 19 children diagnosed as having Autism(via a CARS or ADOS test) and analyzed for the presence of pathogens.The results of this study are shown in FIG. 4. As shown, the stools ofeach of the 19 children diagnosed as having Autism tested positive forvarious pathogens including as H. pylori, Cryptosporidium, E.hystolytica, Giardia, Rotavirus, Camphylobacter, and/or C difficile.

The results of these case studies indicate that there are correlationsbetween the development of various disorders (such as Autism,Parkinson's, ADD and ADHD) and the presence of pathogens and/orcorresponding antigens in an individual's digestive tract. It ispostulated that these pathogens and/or corresponding antigens eitherpromote gastrointestinal dysfunction or have some other direct orindirect effect on the individual, thereby causing such disorders.Further, it is possible that certain mechanisms associated with suchdisorders can be the cause of a proliferation of one or more pathogensin the gastrointestinal tract of an individual. Again, it is to beunderstood that nothing therein shall be taken as a limitation upon theoverall scope of the invention.

For instance, although Case Study 4 involves Autism, based on thecorrelations described herein, it is believed that the present inventionmay be implemented for aiding in the diagnosis of other various PDDssuch as Aspergers syndrome and other related disorders. Furthermore,although Case Study 1 involves Familial Dysautonomia, based on thecorrelations described herein, it is believed that the present inventionmay be implemented for aiding in the diagnosis of various dysautonomicdisorders and dysautonomic conditions, including, but not limited to,Familial Dysautonomia (or Riley-Day Syndrome), Guillaine-Barre Syndrome(GBS) (acute idiopathic polyneuorpathy), fetal fatal insomnia (FFI),diabetic cardiovascular neuropathy, Hereditary Sensory and autonomicnueropathy type III (HSAN III), central autonomic disorders includingmultiple system atrophy (Shy-Drager syndrome), orthostatic intolerancesyndrome including mitral value prolapse, postural tachycardia syndrome(POTS), and idiopathic hypovolemia, dysautonomic syndromes and disordersof the catecholemine family including baroreflex failure,dopamine-B-Hydroxylase deficiency, pheochromocytoma, chemodectina,familial paraganglioma syndrome, tetrahydrobiopterin deficiency,aromatic-L-amino acid decarboxylase deficiency, Menke's disease,monoamine oxidase deficiency states, and other disorders of dopaminemetabolism, dysautonomic syndromes and disorders of the cardiovasularsystem, Chaga's disease, Diabetic autonomic failure, pure autonomicfailure, syncope, hypertension, cardiovascular disease, renal diseaseand SIDS. Further, the present invention is believed to be efficaciousfor diagnosing other neurological disorders such as OCD (obsessivecompulsive disorder) and Tourette's syndrome.

In summary, a method according to the present invention for aiding inthe diagnosis of a disorder comprises analyzing stool samples of anindividual to determine the presence of one or more pathogens including,but not limited to, H. pylori, Cryptosporidium, Entamoeba histolytica,Giardia, Rotavirus, Camphylobacter, and/or C. difficile. Other pathogensthat may be analyzed include, for example, Adenovirus, Cyclospora,Microsoridia, and/or isospora belli. In a preferred embodiment, thepresence of one or more pathogens is determined by a stool immunoassayto determine the presence of associated antigens. The presence of one ormore pathogens comprises a biological marker for determining if anindividual, especially a child, may either have or develop a disorder,including, but not limited to, PDD (such as Autism), Dysautonomia (orother dysautonomic conditions), Parkinson's disease, SIDS, or otherdysautonomic and/or neurological disorders.

Although illustrative embodiments have been described herein withreference to the accompanying drawings, it is to be understood that thepresent invention is not limited to those precise embodiments, and thatvarious other changes and modifications may be to effected therein byone skilled in the art without departing from the scope or spirit of theinvention. All such changes and modifications are intended to beincluded within the scope of the invention as defined by the appendedclaims.

What is claimed is:
 1. A method for determining if an individual has, orcan develop, a disorder or condition, comprising the steps of: obtaininga stool sample from the individual; analyzing the stool sample todetermine the presence of a pathogen; and correlating the presence of apathogen with a disorder or lack thereof.
 2. The method of claim 1,wherein the step of analyzing comprises performing a stool immunoassayto determine the presence of an antigen associated with a pathogen. 3.The method of claim 1, wherein the disorder comprises a PDD (pervasivedevelopment disorder).
 4. The method of claim 1, wherein the disordercomprises a dysautonomic disorder.
 5. The method of claim 1, wherein thedisorder comprises Parkinson's disease.
 6. The method of claim 1,wherein the disorder comprises a neurological disorder.
 7. The method ofclaim 1, wherein the pathogen comprises Helicobacter pylori.
 8. Themethod of claim 1, wherein the pathogen comprises Clostridium difficile.9. The method of claim 1, wherein the pathogen comprisesCryptosporidium.
 10. The method of claim 1, wherein the pathogencomprises Giardia.
 11. The method of claim 1, wherein the pathogencomprises Rotavirus or Adenovirus.
 12. The method of claim 1, whereinthe pathogen comprises Entamoeba histolytica.
 13. A biological markerfor determining if an individual has, or can develop, a disorder orcondition, the biological marker comprising a pathogen in a stool sampleof the individual.
 14. The biological marker of claim 13, wherein thepresence of the pathogen in the stool sample is determined using stoolimmunoassay.
 15. The biological marker of claim 13, wherein the pathogencomprises Helicobacter pylori.
 16. The biological marker of claim 13,wherein the pathogen comprises Clostridium difficile.
 17. The biologicalmarker of claim 13, wherein the pathogen comprises Cryptosporidium. 18.The biological marker of claim 13, wherein the pathogen comprisesGiardia.
 19. The biological marker of claim 13, wherein the pathogencomprises Rotavirus or Adenovirus.
 20. The biological marker of claim13, wherein the pathogen comprises Entamoeba histolytica.